Group B streptococcus is the leading cause of life threatening bacterial infections in neonates and is an increasingly important pathogen among adults with underlying chronic infections. While antibodies to the capsular polysaccharide effectively protect newborns against infection, immunity is type-specific, and at least five antigenically distinct capsular types are important causes of infection and immunity. The goal of this proposal is to elucidate the molecular mechanisms underlying the diversity of capsule structures in order to understand both the regulatory mechanisms controlling capsule expression and the basis for the emergence of new capsule types. Previous studies have identified a 20 kB region of the chromosome that is important in synthesis of the type III capsule. However, despite having identified a number of genes likely to be involved in type III biosynthesis, assignment of functions to these genes has been difficult due to the lack of sequence similarity for some genes, the non-specific nature of the acapsular phenotype resulting from insertion mutations and the inability to demonstrate enzyme activity of gene products expressed in E. coli. Consequently, specific functions have been assigned to only two genes. Because the type III, Ia, and Ib polysaccharides contain identical sugars but have unique glycosidic linkages, the glycosyl transferases responsible for creating these linkages are expected to be encoded by genes unique to the respective loci. Similarly, linkages present in each serotype are expected to involve a common glycosyl transferase and gene. For the present studies, Dr. Wessels proposes to take advantage of the structural relatedness of the capsular polysaccharides of the different serotypes to help elucidate the functions of the capsule genes. Cloning and sequence analysis will be used to define the type Ia and Ib capsule regions, and the architecture and sequence of the different loci will be compared in order to identify conserved genes encoding proteins common to all serotypes, as well as unique genes encoding the serotype-specific glycosyl transferases. Precise functions of the putative glycosyl transferases will be determined by constructing strains containing non-polar knockout mutations in specific genes and determining the structures of the resulting polysaccharides. Site-specific mutagenesis will be used to identify domains of the glycosyl transferases that are responsible for glycosidic bond specificity. Some of the genes conserved across serotypes are expected to function in polymerization and export of the polysaccharides. Stains containing mutations in these genes will be used in in vitro capsule biosynthesis assays to test these predicted functions. These studies are expected to advance the understanding of the molecular basis for production of a major virulence factor in group B streptococcus, and to potentially suggest targets for new pharmacologic therapies.